A Zundel ion in the catalytic proton transfer pathway of [FeFe]-hydrogenase

[FeFe]-hydrogenases are metalloenzymes that catalyze the interconversion of protons, electrons, and molecular hydrogen (H2). Their active site cofactor consists of a [4Fe-4S] cluster ([4Fe]H) and a diiron site ([2Fe]H), forming the so-called H-cluster. In this work, the putative regulatory proton transfer pathway (PTP) toward the [4Fe]H cluster of [FeFe]-hydrogenase CpI from Clostridium pasteurianum is characterized by X-ray crystallography, infrared spectroscopy, and quantum mechanical (QM) calculations. The trajectory consists of asparagine N160, glutamine Q195, and several protein-bound water molecules that might function as a PTP toward cysteine C499 at the [4Fe]H cluster. We have hypothesized that protonation of C499 determines the H-cluster intermediate HoxH (M. Senger et al., Phys. Chem. Chem. Phys., 2018, 20, 3128-3140). The crystal structures of protein variants N160L and Q195L now confirm that the putative regulatory PTP is disrupted. However, infrared spectroscopy reveals that all variants accumulate the HoxH state in a manner comparable to wild-type CpI. In contrast, the CpI variant E279D - previously shown to target the catalytic PTP toward [2Fe]H - is found to enrich the HoxH state independently of reducing agents. This indicates that the determinants of HoxH are located in the catalytic PTP, which emphasizes the importance of HoxH during catalysis and provides evidence against any involvement of the putative regulatory PTP in hydrogen turnover. Supported by QM calculations, a model is proposed in which a conserved water cluster adjacent to E279 is protonated to form a Zundel ion (H5O2+). Our results paint a new picture of the H-cluster in the HoxH state and yield important insight into the catalytic mechanism of [FeFe]-hydrogenases.